Generation of monoclonal antibody producing human hybridomas: Optimized conditions

B. Switzer, I. Blanco, M. Singhai, D. Colcher, D. R. Johnson

Research output: Contribution to journalArticlepeer-review


Human monoclonal antibodies (Mabs) can be created from peripheral blood lymphocytes (PBL) by EBV-transformation (EBV-t) or by direct fusion to human or mouse myeloma lines. EBV-t cell lines are difficult to clone and have low levels of antibody production. As an alternative, we evaluated the development of human Mab producing hybridomas from EBV-t and mitogen stimulated PBL. EBV-t or pokeweed mitogen stimulated (PWM-s) PBL from immunized patients enrolled in a vaccine trial were fused using polyethylene glycol (PEG) at a 1:1 ratio with K6H6/B5 cells. The cells were then plated at 5xl04 per well in RPMI-1640 media (+15% FBS+ 1% ORIGEN HCF (IGEN)), followed by HAT/ouabain selection. High fusion growth rates (avg. 317 wells/107 PBL) were observed. PWM-s hybrids initially produced a more diverse distribution of immunoglobulin classes (IgG and IgM) than EBV-t hybrids EBV-t hybrids maintained antibody production during serial cloning for 120 days. After the last cloning, Mab levels decreased gradually over a period of 60 days, from 7.0-29.0 ug/ml/106 cells to 2.7-16.0 ug/ml/IO6 cells, without losing antigen specificity. We successfully produced and maintained human Mabs with either stimulation method.

Original languageEnglish (US)
Pages (from-to)A1058
JournalFASEB Journal
Issue number6
StatePublished - 1996

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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